Sorbitol mediates age-dependent changes in apple plant growth strategy through gibberellin signaling

Abstract Plants experience various age-dependent changes during juvenile to adult vegetative phase. However, the regulatory mechanisms orchestrating the changes remain largely unknown in apple (Malus domestica). This study showed that tissue-cultured apple plants at juvenile, transition, and adult phase exhibit age-dependent changes in their plant growth, photosynthetic performance, hormone levels, and carbon distribution. Moreover, this study identified an age-dependent gene, sorbitol dehydrogenase (MdSDH1), a key enzyme for sorbitol catabolism, highly expressed in the juvenile phase in apple. Silencing MdSDH1 in apple significantly decreased the plant growth and GA3 levels. However, exogenous GA3 rescued the reduced plant growth phenotype of TRV-MdSDH1. Biochemical analysis revealed that MdSPL1 interacts with MdWRKY24 and synergistically enhance the repression of MdSPL1 and MdWRKY24 on MdSDH1, thereby promoting sorbitol accumulation during vegetative phase change. Exogenous sorbitol application indicated that sorbitol promotes the transcription of MdSPL1 and MdWRKY24. Notably, MdSPL1-MdWRKY24 module functions as key repressor to regulate GA-responsive gene, Gibberellic Acid-Stimulated Arabidopsis (MdGASA1) expression, thereby leading to a shift from the quick to the slow-growth strategy. These results reveal the pivotal role of sorbitol in controlling apple plant growth, thereby improving our understanding of vegetative phase change in apple.


Introduction
Many perennial woody plants experience a long juvenile phase before f lowering [1][2][3].The transition from the juvenile to the adult phase, referred to as the vegetative phase change, is highly regulated by various endogenous cues such as plant age, sugars, and phytohormones [4,5].Vegetative phase change of plants leads to a series of morphology and physiology changes, including leaf morphology, photosynthetic traits, sink-source balances, leaf hormone dynamics, and growth strategies [5][6][7][8].For example, changes in the leaf shape and trichome appearance of leaves are the most commonly used markers of vegetative phase change in Arabidopsis [7,8].In Myrtaceae (Eucalyptus globulus ssp.globulus), vegetative phase change is accompanied by an increase in the leaf cuticle thickness, stomatal density, and the size of leaf blade and the disappearance of epicuticular wax in the leaves [9].Juvenile Populus tremula x alba leaves also have a higher leaf nitrogen, specific leaf area (SLA), and mean mass-based photosynthetic rates than adult leaves [10].SLA, the ratio of leaf area to dry mass, is determined by the thickness of the leaf blade and cell density; thus, a high SLA contributes to the photosynthetic performance of the plants [10,11].Recent studies have shown that leaf photosynthetic changes alter plant growth and cause a switch from the fast-to the slow-growth strategy during the vegetative phases of Arabidopsis thaliana, Zea mays, and P. tremula x alba [12].However, the regulatory mechanisms underlying age-related changes in the growth strategies of perennial woody plants remain unclear.
Sugars are also key age-dependent internal signals for vegetative phase change [19,20].Sucrose, glucose, and fructose promote vegetative phase change by suppressing MIR156A/C expression [20,21].Arabidopsis chlorophyll-deficient mutant chlorina1-4 (ch1-4) reduces the photosynthetic rate and delays vegetative phase change, but exogenous glucose can restore this phenotype [19,21].Moreover, plant sucrose levels regulate the timing of Arabidopsis vegetative phase change via the trehalose 6-phosphate (T6P) pathway [22].TREHALOSE PHOSPHATE SYNTHASE1 (TPS1) is a key enzyme in T6P synthesis.Thus, the loss of TPS1 increases MIR156A/C expression and prolongs the juvenile phase in Arabidopsis [22,23].In the Rosaceae family, sorbitol is a special photosynthetic product in the leaves of many fruit trees, and it can be transformed into glucose and fructose by sorbitol dehydrogenase (SDH) [24][25][26].Several studies have demonstrated that sorbitol is a key signal regulating f lower bud formation in loquat (Eriobotrya japonica) [27], pollen tube growth [28], and resistance against Alternaria alternata in apple [29].However, the role of sorbitol in regulating vegetative phase change in fruit trees remains unknown.
Thus, this study showed that the vegetative phase change in apple contributes to age-dependent changes in plant growth, photosynthetic performance, hormone levels, and carbon distribution.RNA-seq analysis revealed that MdSDH1, MdGASA1, MdSPL1, and MdWRKY24 play critical roles in the transition from juvenile to adult phase.Further studies showed that MdSDH1 is important in regulating sorbitol levels via the MdSPL1-MdWRKY24 module.Moreover, sorbitol and MdSPL1-MdWRKY24 module form a feedback loop that regulates the expression of the GA response gene, MdGASA1, thus modulating the transition of growth strategies during vegetative phase change in apple.Our results provide new insights into the regulatory mechanism by which sorbitolmediated growth transitions in apple.

Plant morphological and leaf physiological age-dependent changes during vegetative phase change in apple
Vegetative phase changes in apple can be divided into three stages (juvenile, transition, and adult) (Fig. 1a).In a previous study, we obtained tissue-cultured apple plants at juvenile, transition, and adult phase and designated 1y, 3y, and 5y.Results showed that the dynamic changes of leaf size, abaxial trichome, epidermal cell size, stomatal density, SLA, and miR156 level in tissuecultured apple plants (1y, 3y, and 5y) were consistent with those in the source tree (different stages) [43].Moreover, we revealed the regulatory mechanism of CK-mediated changes in leaf size during vegetative phase change [43].To further investigate how vegetative phase change contributes to the growth strategies changes in apple, we compared the plant morphology across 1y, 3y, and 5y apple plants.The results showed an obvious decrease in plant height and internode number from 1y to 5y plants, suggesting a shift from the quick to the slow-growth strategy during the vegetative phase change (Fig. 1a-c).The net photosynthetic rate (Pn), chlorophyll (Chl) content, SLA, leaf nitrogen (leaf N), maximal photochemical efficiency (Fv/Fm), and regulatory energy dissipation (Y(NPQ)) continuously decreased from 1y to 5y plants (Fig. 1d-i), consistent with the phenotype of the 1y, 3y, and 5y apple plants.
Moreover, the contents of four hormones (abscisic acid, ABA; gibberellin 3, GA3; auxin, IAA; 1-aminocyclopropanecarboxylic acid, ACC) in 1y, 3y, and 5y apple plants were measured.The content of ABA, IAA, and ACC gradually increased from 1y to 5y plants (Fig. 1j-l), suggesting that they play positive roles in the transition from juvenile to adult phase in apple.In contrast, the GA3 content gradually decreased from 1y to 5y plants.The GA3 content of 1y plants were 1.89-fold and 3.08-fold higher than in 3y and 5y plants, respectively (Fig. 1m).

Leaf carbon distribution age-dependent changes during vegetative phase change in apple
We further measured the contents of sugars and amino acids in 1y, 3y, and 5y apple plants by targeted metabolomics.The contents of glucose, starch, and 12 amino acids (alanine, aspartate, arginine, histidine, isoleucine, leucine, glutamate, lysine, proline, serine, valine, and glycine) gradually decreased from 1y to 5y plants (Fig. 2a, b, e-p).On the contrary, the contents of sucrose and sorbitol gradually increased from 1y to 5y plants (Fig. 2c and d), suggesting that they play positive roles in the transition from juvenile to adult phase in apple.These results demonstrate that the photosynthetic products in 1y plants may be used for growth but accumulated as sugars in 5y plants.Moreover, 60-80% of the photosynthates in apple leaves were sorbitol [44], with approximately 19-fold higher contents than sucrose (Fig. 2c and d), suggesting that sorbitol is the major carbon source during apple growth.Thus, sorbitol might participate in the transition from the quick to the slow-growth strategy in apple.

RNA-seq data shows that MdSDH1 is related to vegetative phase change
To identify genes associated with age-dependent changes in growth strategy during vegetative phase change, we performed an RNA-Seq analysis in the top fifth or sixth fully expanded leaves of 1y, 3y, and 5y apple plants.The three RNA-Seq biological replicates had high Pearson correlation analysis, indicating the reliability of sequencing data (Fig. S1, see online supplementary material).By comparing the gene expression levels, 211, 648, and 560 differentially expressed genes (DEGs) were identified in the comparisons of 1y vs. 3y, 1y vs. 5y, and 3y vs. 5y, respectively (Fig. S2a, see online supplementary material).The DEGs enriched the following GO terms: oxidation-reduction process (GO: 0055114), carbohydrate binding (GO: 0030246), energy reserve metabolic process (GO: 0005975), carbohydrate metabolic process (GO: 0005975), and other biological processes (Fig. 3a).Further, the DEGs in 1y vs 3y vs 5y comparisons significantly enriched the following KEGG pathways: carbohydrate metabolism, amino acid metabolism, and biosynthesis of other secondary metabolites (Fig. S2b, see online supplementary material), including 'starch and sucrose metabolism', 'phenylpropanoid biosynthesis', and 'tyrosine metabolism' (Fig. 3b).

MdSDH1 regulates plant growth by altering endogenous GA3 levels in apple
The full-length coding sequence of MdSDH1 is 1107 bp and encodes a protein with 368 amino acids.Reverse transcriptionquantitative PCR (RT-qPCR) showed that MdSDH1 is expressed in all tested apple tissues, but the expression is highest in apple leaves (Fig. 4a).An expression analysis revealed that lowlight treatment significantly down-regulated MdSDH1 expression (Fig. 4b), indicating that MdSDH1 is responsive to environmental signals in apple plants.We further fed the youngest fully expanded leaves from 30-day-old seedlings with 50 mM sorbitol for 12 h and examined MdSDH1 transcript levels and SDH enzyme activity at different time points.Exogenous sorbitol strongly induced MdSDH1 transcript levels and SDH enzyme activity (Fig. 4c and d), suggesting that MdSDH1 is a key enzyme for sorbitol catabolism in apple.
We constructed silencing vectors in apple plants via virusinduced gene silencing (VIGS) assays to analyse the function of MdSDH1 in apple.RT-qPCR analysis showed that MdSDH1 transcripts in TRV-MdSDH1 plants were significantly reduced by 75% compared with TRV plants (Fig. 4g).Additionally, MdSDH1 silencing significantly reduced SDH enzyme activity and promoted sorbitol accumulation in the TRV-MdSDH1 apple plants (Fig. 4h and i), demonstrating that the SDH enzyme governs sorbitol catabolism.Phenotypic analysis revealed that TRV plants were taller than TRV-MdSDH1 plants after 30 days (Fig. 4e, f, and j).At the same time, the numbers and lengths of internodes, which primarily determine the plant height, were significantly lower in TRV-MdSDH1 plants than in TRV plants (Fig. 4 k and l).These results demonstrate that silencing MdSDH1 inhibits apple plant growth.Besides, exogenous sorbitol application significantly reduced the plant height and internode length (Fig. S3a-d, see online supplementary material), suggesting that sorbitol accumulation significantly inhibits apple plant growth.This result was consistent with the previous finding that the growth ability in 1y apple plants was stronger than that 5y apple plants when sorbitol accumulated in 5y apple plants (Figs 1b  and 2d).
Various endogenous hormones affect plant growth; we thus measured the GA3, IAA, and cytokinin trans-zeatin (tZ) levels in TRV and TRV-MdSDH1 plants.Compared with TRV plants, the contents of IAA and tZ were insignificantly different, while the GA3 content was significantly lower in TRV-MdSDH1 (Fig. 4m-o).To further confirm whether the TRV-MdSDH1 phenotype is caused by GA3 deficiency, we treated TRV-MdSDH1 plant with 10 μM exogenous GA3.The GA3 application promoted the growth of TRV-MdSDH1 apple plants (Fig. S4a-d, see online supplementary material), indicating that MdSDH1 silencing decreased the GA3 content, thereby repressing the growth of apple.

MdSDH1 is directly regulated by MdSPL1 and MdWRKY24
To further study the mechanism for MdSDH1 during vegetative phase change, we used the MdSDH1 promoter as the bait in a yeast-one hybrid (Y1H) system to screen apple cDNA library.The results showed that a SBP (SPL) TF, (MdSPL1), and a WRKY TF (MdWRKY24) binds to the MdSDH1 promoter in yeast cells (Fig. 5a and b).Previous studies showed that SPLs and WRKY TFs are important in regulating plant growth [34,37,45,46].In this study, an analysis by RT-qPCR showed that the transcript levels of MdSPL1 and MdWRKY24 were linearly increasing from 1y to 5y plants (Fig. S5a and b, see online supplementary material), suggesting that they are age-dependent TFs.Our previous studied demonstrated that CK regulates age-mediated changes in leaf size through the mdm-miR156a/MdSPL14 module regulate in apple [43].Sequence complementarity analysis showed that no binding region of mdm-miR156a was detected on MdSPL1 mRNA (Fig. S6a, see online supplementary material).Moreover, no decrease in the expression of MdSPL1 was detected when the mdm-miR156a was overexpressed in apple leaves via transient infiltration (Fig. S6b, see online supplementary material).To further verify the mdm-miR156a on MdSPL1 activity, a dual luciferase-based miRNA sensor assay was conducted in tobacco leaves.There was no decrease in f luorescence signal and the relative Luciferase/Renilla (LUC/REN) activity with the coexpression of mdm-miR156a and MdSPL14 compared with the control group (Fig. S6c and d, see online supplementary material).Therefore, these results demonstrate that mdm-miR156a is unable to directly target MdSPL1 in apple.
The dual-luciferase (LUC) assay revealed that expressing MdSPL1 and MdWRKY24 in Nicotiana benthamiana leaves significantly decreased the relative LUC/REN activity driven by the MdSDH1 promoter (Fig. 5c-d), suggesting that MdSPL1 and MdWRKY24 repressed MdSDH1 transcription.The electrophoretic mobility shift assay (EMSA) showed that MdSPL1 and MdWRKY24 can bind directly to the GTAC sites and W-box in the MdSDH1 promoter, respectively (Fig. 5e).The ability of MdSPL1 and MdWRKY24 to bind the GTAC sites and W-box in the MdSDH1 promoter region gradually decreased as the amount of competitor probes increased.After the core base of the W-box and GTAC sites mutated, MdWRKY24 and MdSPL1 cannot bind to the mutant probe (Fig. 5e).These findings reveal that MdSPL1 and MdWRKY24 function as MdSDH1 transcriptional inhibitors.

The MdSPL1-MdWRKY24 module promotes sorbitol accumulation by repressing MdSDH1 expression
An assessment of whether MdSPL1 physically interacted with MdWRKY24 by yeast-two hybrid (Y2H) assay revealed that the yeast strains with co-expressed MdSPL1 and MdWRKY24 had normal growth on the -T/−L/-H/−A media (Fig. 6a).To further validate the interaction of the two proteins, an in vivo bimolecular f luorescence complementation (BiFC) assay was conducted.The result showed that a yellow f luorescence signal was observed in the nucleus and cell membranes when MdSPL1-cYFP and MdWRKY24-nYFP were co-transformed in N. benthamiana leaves (Fig. 6b).A pull-down assay using MdSPL1-His and MdWRKY24-GSH protein revealed that MdSPL1 and MdWRKY24 interact in vitro (Fig. 6c).Furthermore, EMSA assays were performed to determine the consequence of the MdSPL1-MdWRKY24 interaction on the transcriptional regulation of MdSDH1.The addition of MdSPL1 protein and MdWRKY24 protein significantly enhanced the binding of both proteins to the biotin probes, respectively (Fig. 6d), indicating that the interaction between MdSPL1 and MdWRK24 increase the binding ability of both MdSPL1 and MdWRK24 proteins on MdSDH1 promoter.A LUC assay on N. benthamiana leaves further showed that the MdSPL1 and MdWRKY24 co-expression significantly repressed the MdSDH1 pro : LUC expression than the effect of MdSPL1 or MdWRKY24 alone (Fig. 6e and f).Therefore, there findings indicate that MdSPL1 interacts with MdWRKY24 and enhance the inhibition of MdSDH1 by both MdSPL1 and MdWRKY24.
To confirm the role of MdSPL1 and MdWRKY24 in regulating endogenous sorbitol levels, MdSPL1-OE, MdWRKY24-OE, and the MdSPL1-OE + MdWRKY24-OE combination were overexpressed in apple leaves via vacuum infiltration.RT-qPCR analysis showed that overexpression of MdSPL1 and MdWRKY24 in apple leaves significantly reduced the transcript levels of MdSDH1 (Fig. S7a and b, see online supplementary material; Fig. 6g).Obviously, co-expressing MdSPL1-OE and MdWRKY24-OE significantly reduced SDH enzyme activity, leading to a higher sorbitol content than in MdSPL1-OE or MdWRKY24-OE alone (Fig. 6h and i).These results demonstrate that the MdSPL1-MdWRKY24 module positively regulates sorbitol levels by repressing MdSDH1 expression in apple.

Sorbitol inhibits MdGASA1 expression via the MdSPL1-MdWRKY24 module
The above results support the viewpoint that sorbitol represses plant growth by affecting the GA3 level, but the role of GA signaling involved in plant growth during the juvenile to adult vegetative phase remains yet to have been studied.RNAseq data further revealed a GA-responsive gene Gibberellic Acid-Stimulated Arabidopsis (MdGASA1) gene that exhibited agedependent expression patterns in apple, with high expression in 1y plants, which gradually decreased during the vegetative phase change (Fig. S8a, see online supplementary material).RT-qPCR analysis showed that GA3 treatment strongly up-regulated MdGASA1 expression, which was down-regulated by sorbitol feeding (Fig. S8b and c, see online supplementary material), indicating that MdGASA1 is a positive regulator of the GA signaling but is a negative regulator of sorbitol signaling in apple.
To identify whether MdGASA1 is directly regulated by MdSPL1 and MdWRKY24, Y1H, LUC and EMSA assays were performed.Y1H assays showed that yeast cells that co-expressed pro-MdGASA1 and pGADT7-MdSPL1/pGADT7-MdWRKY24 grew normally on -Leu/-Ura media with 150 mmol/L of Aureobasidin (AbA) (Fig. 7b), suggesting that MdSPL1 and MdWRKY24 can bind directly to the MdGASA1 promoter.LUC assay revealed that expressing MdSPL1 and MdWRKY24 in N. benthamiana leaves significantly decreased the relative LUC/REN activity driven by the MdGASA1 promoter (Fig. 7c and d), suggesting that MdSPL1 and MdWRKY24 repress MdGASA1 transcriptional activity.EMSA further showed that MdSPL1 and MdWRKY24 can bind directly to the GTAC sites and W-box in the MdGASA1 promoter in vitro, respectively (Fig. 7e).These findings reveal that MdSPL1 and MdWRKY24 function as MdGASA1 transcriptional inhibitors, which was consistent with the previous result that GA3 gradually decreases during vegetative phase change (Fig. 1m).
Furthermore, EMSA assays were performed to determine the consequence of the MdSPL1-MdWRKY24 interaction on the transcriptional regulation of MdGASA1.The addition of MdSPL1 protein and MdWRKY24 protein significantly enhanced the binding of both proteins to the biotin probes, respectively (Fig. 7f), indicating that the interaction between MdSPL1 and MdWRK24 increase the binding ability of both MdSPL1 and MdWRK24 proteins on MdGASA1 promoter.LUC assay on N. benthamiana leaves further confirmed that the MdSPL1 and MdWRKY24 co-expression significantly decreased the MdGASA1 pro : LUC expression than the effect of MdSPL1 or MdWRKY24 alone (Fig. 7g), suggesting that MdSPL1 interacts with MdWRKY24 and enhances the inhibition of MdGASA1 by both MdSPL1 and MdWRKY24.These results demonstrate that MdSPL1 and MdWRKY24 coordinately repress MdGASA1 transcription.
A previous study has shown that sorbitol plays a key signaling role in by regulating the expression of TFs, thereby controlling downstream target genes in apple [47].Exogenous sorbitol application in apple leaves was conducted to determine whether sorbitol signaling is involved in MdGASA1 transcription by affecting the MdSPL1-MdWRKY24 module.The results showed that exogenous sorbitol significantly induced MdSPL1 and MdWRKY24 transcript levels in apple leaves (Fig. S9a and b, see online supplementary material), indicating a feedback loop between sorbitol and the MdSPL1-MdWRKY24 module.These results indicate that sorbitol inhibits the expression of MdGASA1 by activating MdSPL1 and MdWRKY24 in apple.

Silencing MdGASA1 decreased plant growth by affecting cell expansion in apple
We generated MdGASA1-silenced plants through VIGS assay to validate the role of MdGASA1 in regulating plant growth.RT-qPCR analysis showed that MdGASA1 transcripts in TRV-MdGASA1 plants were significantly reduced by 72% compared with TRV plants (Fig. 8b).Phenotypic analysis showed that TRV plants had higher plant heights than the TRV-MdGASA1 plants after 30 days (Fig. 8a and c).At the same time, the number and length of internodes were significantly higher in TRV than TRV-MdGASA1 plants (Fig. 8d and e), implying that MdGASA1 inhibits apple plant growth by shortening the internode length.Cytological observations of transverse stems showed that TRV plants had a significantly higher cortex cell length and cortex cell area than TRV-MdGASA1 plants, but the xylem size was insignificantly different (Fig. 8f-i).Similarly, the longitudinal stem cells were larger in TRV plants than in TRV-MdGASA1 plants, and the cell numbers were higher in TRV plants than in TRV-MdGASA1 plants (Fig. 8j-m).These results demonstrate that silencing MdGASA1 suppresses cell expansion along the longitudinal axis, shortening the internode length.

Discussion
Vegetative phase change is a complex process in perennial woody plants [1][2][3][4].Plants experience various age-dependent changes during this process, including morphological changes, photosynthetic traits, and growth strategies [1,[48][49][50].However, changes in leaf sink-source balances, hormone dynamics, and abiotic stress responses are usually overlooked in apple plant research due to the limitations of the long juvenile phase.Thus, the relationship between these age-dependent changes and plant morphology in apple has remained unclear.This study obtained tissuecultured apple plants to reveal the changes in plant growth, photosynthetic capacity, hormone levels, and carbon distribution during vegetative phase change.The study further provided detailed mechanistic evidence of the role of sorbitol in controlling the shifts from quick to slow-growth strategy in apple.The results highlight the function of sorbitol in connecting GA

Sorbitol is essential for the transition from quick to slow-growth strategy in apple
Sugars, including sucrose, glucose, and T6P, are a carbon source and key signaling molecules controlling the vegetative phase change in plants [5,6,51,52].Previous studies have shown that photosynthetic sucrose is not utilized directly; cytosolic invertase (CINV) irreversibly catalyzes its conversion to glucose and fructose, providing an essential carbon source for plant growth [53].In Arabidopsis, sucrose-induced PAP1 TF increases the transcription of SPL9 by directly binding to its promoter, which triggers the sucrose-mediated vegetative phase change through the miR156A/SPL9 module [54].These results indicate that sugar accumulation in the leaves potentially functions as an age-dependent signal that regulates the downstream gene expression, contributing to vegetative phase change [5,21].Sorbitol is the primary photosynthetic product in Rosaceae fruit trees.In contrast, sucrose is the main photosynthate in the other plants [26,27,55] and is closely related to growth, development, and stress resistance [24,26,48].
In this study, sorbitol and MdSDH1 exhibited an age-dependent pattern.MdSDH1 was highly expressed in 1y plants, and sorbitol massively accumulated in 5y plants (Figs 2d and 3d).Moreover, the content of 12 amino acids gradually decreased during the vegetative phase change (Fig. 2e-p).Therefore, 1y plants possibly have strong sinks for protein synthesis and provide a steady carbon supply for energy production [4].These results indicate that sorbitol mainly functions as a carbon source for the juvenile phase and a signal-modulating phase transition in the adult phase.Additionally, the MdSDH1-silenced phenotype of apple plants revealed that sorbitol accumulation suppresses growth (carbon consumption), consistent with the changes of growth in 1y, 3y, and 5y apple plants.Therefore, sorbitol is a candidate marker for vegetative phase change, and MdSDH1 is likely the key hub gene for regulating sorbitol-mediated shifts from quick to slow-growth strategy during juvenile to adult vegetative phase in apple.
In Arabidopsis, SPL10 regulates the age-mediated vegetative phase change by interacting with WRKY12 and WRKY13 [56], indicating that SPLs and WRKYs TF are critical in the aging pathway.Interestingly, several WRKY TFs function as negative regulators of plant height by decreasing brassinosteroid (BR) production or repressing the expression of cell elongation genes [31,37,38,47].The results of this study revealed that MdSPL1 and MdWRKY24 target MdSDH1, synergistically suppressing the expression of MdSDH1 by binding to its promoter (Fig. 5).At the same time, overexpressing MdSPL1 and MdWRKY24 in apple leaves decreased the SDH enzyme activity and promoted sorbitol accumulation (Fig. 6h and i).These findings demonstrate that the age-mediated MdSPL1-MdWRKT24 module represses apple plant growth by promoting sorbitol accumulation.The exogenous sorbitol spraying test further confirmed this point (Fig. S3, see online supplementary material).

MdGASA1 is involved in sorbitol-mediated growth transition via the MdSPL1-MdWRKY24 module in apple
Sorbitol also functions as a signaling substance that regulates plant growth and development.During f lower bud formation in loquat, sorbitol promotes hyperoside biosynthesis by activating the transcription of EjERF12 and the MADS-box TF family gene, EjCAL [27].In apple, sorbitol regulates downstream developmental genes by activating a key TF, MYB39L [47].Suppressing MdMYB39L expression in apple pollen results in stamen development and reduces pollen tube growth, and this phenotype can be partially restored by exogenous sorbitol application during f lower development [47].Previous studies have shown that sorbitol controls sugar transport into the pollen tube by regulating the expression of pollen tubule transporter HT1.7, thus promoting pollen tube growth in apple [28].In this study, sorbitol significantly activated the expression of two age-related TF genes, MdSPL1 and MdWRKY24.MdSPL1 interacts with MdWRKY24 to synergistically suppress MdSDH1 expression (Fig. 6; Fig. S9, see online supplementary material), suggesting that sorbitol-induced MdSPL1 and MdWRKY24 transcription via a feedback loop.Moreover, MdSPL1 and MdWRKY24 act synergistically to inhibit MdGASA1 expression, thus reducing GA signaling responses (Fig. 7).A recent study revealed that overexpressing OsWRKY36 results in dwarfness in wheat due to the increased SLENDER RICE 1 (SLR1) transcription and the suppression of GA signaling [38].These results provide evidence that sorbitol is a signal that regulates GA responses by activating age-related TFs during vegetative phase change.
Gibberellin is important in determining plant height [40,41,57].The miR166 target gene THB14-LIKE controls plant height by directly repressing GA biosynthesis genes (GmGA1 and GmGA2) expression and activating GA catabolic gene GIBBERLLIN 2 OXI-DASE 2 (GmGA2ox2) expression in soybean [58].The GASA family genes are key downstream response genes in the GA signaling pathway, whose expression is strongly induced by GA3 and functions as a positive regulator for plant growth and development [59,60].In Arabidopsis, AtGASA6 regulates seed germination and hypocotyl elongation by integrating GAs, ABA, and glucose signaling [61].In soybean, GmGBP1 and GmGAMYB interaction induces the GA signal to increase plant height by activating the GmSAUR bound to the GmGASA32 promoters [62].Overexpressing GmGASA32 promotes plant growth by interacting with GmCDC25 [63].These results demonstrate that silencing MdGASA1 represses apple plant growth by reducing the elongation of shoot stem cells (Fig. 8).
In conclusion, this study provided detailed phenotypic and physiological evidence demonstrating that sorbitol regulates agedependent changes in growth strategies for apple through the MdSPL1-MdWRKY24 module during vegetative phase change.Therefore, we propose a model for sorbitol-mediated changes in growth strategies from the juvenile to the adult vegetative phase (Fig. 9).During juvenile to adult vegetative phase, the age-mediated MdSPL1-MdWRKY24 module facilitates sorbitol accumulation by repressing MdSDH1 transcription, thus reducing the carbon source supply and increasing the sorbitol signal.Simultaneously, the increased sorbitol signaling promotes MdSPL1-MdWRKY24 expression via a feedback loop, thereby repressing MdGASA1 transcription and decreasing apple plant growth in the adult phase.

Plant materials and growth conditions
The F1 progeny of 'Ambrosia × Honeycrisp' were grown in a nutrient bowl and placed in solar greenhouses (25 • C, 16/8 h light/dark) at Northwest A&F University, Yangling (34 • 20 N, 108 • 24 E), Shaanxi Province, China in 2015.In mid-March 2018, 16 plants with branches were selected and planted in fields to grow.In 2020, we selected one apple tree with a short juvenile period from 16 five-year-old apple trees and named as the source tree.In the source tree, 1-, 3-, and 5-year-old branch represents juvenile, transition, and adult phases, respectively.Thus, the shoot tips were collected from the 1-, 3-, and 5-year-old branch of the source tree and obtained tissue-cultured apple plants at juvenile, transition, and adult phase via in vitro tissue culture [64], and named 1y, 3y, and 5y, respectively (Fig. 1a).The tissue-cultured apple plants were cultured on rooting media for 40 days and transferred into plastic pots (6 × 6 cm) with matrix soil of organic substrate/perlite/vermiculite (3:1:1) and grown in a light incubator.After 50 days, the plants were transplanted into plastic pots (30 × 18 cm) filled with the soil/organic matter (v:v, 5:1) mixture and placed in a greenhouse to grow.

Physiological measurements and morphological observation
Plant height measurements were taken from four-month-old apple plants.The top fifth or sixth fully expanded leaves were used for physiological measurements.Leaf N content was detected in the dried samples using an AA3 continuous f low analyzer (SEAL, Germany).After leaf area was measured using a leaf area scanner (Perfection V19, EPSON, Nagano, Japan), the samples were dried in an oven at 65 • C until constant weight.The SLA was calculated using the following formula: SLA = dry weight/leaf area.Chlorophyll was extracted for 24 h using 80% acetone, and the chlorophyll content was determined using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 645 and 663 nm [65].Photosynthetic parameters were monitored using a CIRAS-3 portable photosynthesis system (CIRAS, MA, USA).Leaves were placed in the chamber (18 mm diameter) at 25 • C, 1000 μmol m −2 s −1 illumination, 500 μmol s −1 airf low rate, and 400 μmol mol −1 CO 2 .The chlorophyll (Chl) f luorescence parameters were determined using a Plant Chlorophyll f luorescence imaging system (Walz, Effeltrich, Germany) after 30 min of exposure to darkness.Histological analysis of stems as described by Yao et al. [66] and a BX63 light microscope (Olympus, Japan) was used to observe the cross sections.ImageJ software (https:// imagej.net/software/imagej/)was used to measure the cell length and area.

RNA extraction and RT-qPCR analysis
Total RNA was extracted from the top fifth or sixth fully expanded leaves of apple plants using a plant RNA extraction kit (FORE-GENE 129710 Co., Ltd, Chengdu, China).RT-qPCR analysis was performed in a LightCycler 96 instrument.The primers used are shown in Table S1 (see online supplementary material).

Determinations of free amino acids and sugars
The top fifth or sixth fully expanded leaves of apple plants were used for determination of amino acids and sugars.Amino acids were extracted as described previously by Huo et al. [67].The sugar contents were measured using the gas chromatographymass spectrometry (GC-MS) system equipped with a DB-5MS column (ISQ & TRACE ISQ, Thermo Fisher Scientific, MA, USA), as previously described by Hu et al. [68].Further, the sorbitol content was measured using test kits from Suzhou Comin Biotechnology test kits following the manufacturer's protocol.

Measurements of the contents of phytohormones
The top fifth or sixth fully expanded leaves of apple plants were used for determination of hormones.Phytohormones were extracted as previously described by Jia et al. [43].The phytohormone contents were measured using a QTRAP5500 HPLC-MS (AB SCIEX, DC, USA) after nitrogen blowing.

Exogenous GA3 and sorbitol treatment
Brief ly, 30-day-old 1y apple plants were grown in plastic 8 × 8 pots and were sprayed with 10 μM GA3.For exogenous sorbitol feeding, leaves from 30-day-old seedlings were fed with exogenous sorbitol as described previously [47].The fully expanded leaves were collected at 0, 1, 3, 6, 9, and 12 h after treatment for RT-qPCR analysis.TRV-MdSDH1 apple plants were sprayed with 10 μM GA3 and 100 μM sorbitol (PH102-X; Coolaber) every 2 days, and the controls were water-treated.The indicators were calculated after 30 d.

Transcriptome sequencing
The top fifth or sixth fully expanded leaves of 1y, 3y, and 5y apple plants were used for RNA-seq.The eukaryotic mRNA was enriched using Oligo (dT) beads.The variations were determined using the RSEM software.Gene Denovo Biotechnology Co (Guangzhou, China) conducted subsequent analyses.

Y1H assay
The MdSDH1 and MdGASA1 promoter fragments (1500 bp) were fused into the pHIS2 vector, and the CDSs of MdSPL1 and MdWRKY24 were fused into the pGADT7 vector.The fusion vectors were transformed into the Y187 yeast.The monoclonal clone selected from the SD/-Leu medium was inoculated to SD/-Leumedium supplemented with Aureobasidin A (AbA) to observe yeast growth.

Y2H assay
The CDS region of MdSPL1 was fused into pGBKT7, and the CDS region of MdWRKY24 was fused into pGADT7.The fusion vectors were co-transformed into yeast strain Y2H Gold.The monoclonal clone growing on the DDO (SD/−Leu/−Trp) medium was inoculated to the selective QDC (SD/−Leu/−Trp/-His/−Ade) and QDC + X-α-Gal medium to observe yeast growth, respectively.

Dual-luciferase reporter assay
The promoter sequences (1500 bp) of MdSDH1 and MdGASA1 were fused into the reporter pGreenII 0800-LUC vector, and the CDS regions of MdSPL1 and MdWRKY24 were fused into the effector pGreenII62-SK vector.Next, specified combinations of the recombinant plasmids were co-expression into N. benthamiana leaves.After 48-60 h of transformation, LUC f luorescence image was observed using an in vivo plant imaging system (PlantView100; Guangzhou Biolight Biotechnology Co., Ltd, Guangzhou, China).The relative LUC/REN activity was detected using a dual-luciferase reporter gene assay kit (Yeasen, Shanghai, China).

EMSA
The MdSPL1-GST and MdWRKY24-GST recombinant vectors were transformed into Escherichia coli Rosseta (DE3), and the proteins were purified using GST beads (Beyotime, Shanghai, China).The EMSA assays were conducted using a Light Shift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).

BiFC assay
The CDS regions of MdSPL1 and MdWRKY24 were cloned into the pSPYCE and pSPYNE vectors, respectively.The recombinant vectors were transformed into Agrobacterium tumefaciens GV3101 and co-expressed in N. benthamiana leaves.The f luorescence was observed using a laser scanning microscope (TCS-SP8 SR; Leica) after 48-60 h of injection.

Pull-down assay
The CDS regions of MdSPL1 and MdWRKY24 were inserted into pET-32a and pGEX-4T-1 vectors, respectively.The recombinant vectors were transformed into E. coli (Rosetta strain) for protein expression.The purified MdWRKY24-GST protein was incubated to anti-GST magnetic beads at 4 • C for 12 h and then the purified protein MdSPL1-His was added and incubated at room temperature for 2 h.A western blot was performed using anti-GST (Beyotime, Shanghai, China) and anti-His antibodies (Yeasen, Shanghai, China), respectively.

Virus-induced gene silencing
A specific 200 bp fragment of MdSDH1 and 185 bp fragment MdGASA1 were inserted into the pTRV2 vector, and the recombinant vectors were transformed into A. tumefaciens strain GV3101.
The agrobacteria that were harbored contained the pTRV2-MdMdSDH1 or pTRV2-MdGASA1, and pTRV1 were cultured to an OD 600 of approximately 1.0.The VIGS assays were conducted as described by Zhu et al. [69].

Statistical analyses
Origin software, version 2020 was used for statistical analysis.The data are shown as values represent means ± SD (standard deviation).The statistical significance was determined by oneway ANOVA, and P < 0.05 was considered statistically significant.

Figure 3 .
Figure 3. RNA-seq identification of MdSDH1 from juvenile to adult vegetative phase in apple.(a) Gene Ontology (GO) enrichment of DEGs.(b) KEGG pathway enrichment analysis of DEGs in the three pairwise comparisons.(c) Proposed metabolism of photosynthates (sugars) in apple plants.(d) The line chart represents the DEGs (FPKM) in 1y, 3y, and 5y apple plants.

Figure 4 .
Figure 4. Silencing MdSDH1 inhibits apple plant growth.(a) Relative expression levels of MdSDH1 in different apple tissues.(b) Expression analysis of MdSDH1 in apple leaves under low-light treatment.(c) Expression analysis of MdSDH1 and (d) SDH enzyme activity in apple leaves under 50 uM sorbitol feeding.(e) and (f) Phenotypes of TRV-MdSDH1 and TRV apple plants.(g) Relative expression level of MdSDH1, (h) SDH enzyme activity, (i) sorbitol content, (j) plant height, (k) internode number, (l) internode length, (m) GA3 content, (n) IAA content, and (o) tZ content in TRV-MdSDH1 and TRV plants.Values represent means ± SD (n = 3).Values with different letters are significantly different based on one-way ANOVA and Tukey's test (P < 0.05).

Figure 5 .
Figure 5. MdSPL1 and MdWRKY24 bind directly to MdSDH1 promoter.(a) Schematic diagram of the MdSDH1 promoter.(b) Y1H assay identify the binding ability of MdSPL1 and MdWRKY24 on MdSDH1 promoter.(c) Schematic diagram of reporter and effector constructs.(d) The images of luciferase and relative LUC/REN activities between MdSDH1 promoter and MdSPL1 and MdWRKY24.(e) EMSA assay of MdSPL1 and MdWRKY24 proteins binding to MdSDH1 promoter.Mut, mutant probes.Values represent means ± SD (n = 3).Values with different letters are significantly different based on one-way ANOVA and Tukey's test (P < 0.05).

Figure 6 .
Figure 6.MdSPL1 interacts with MdWRKY24 to coordinately repress MdSDH1 expression.(a) A Y2H assay shows the interaction of MdSPL1 and MdWRKY24 in yeast cells.(b) BiFC assay shows that the interaction of MdSPL1 and MdWRKY24 in Nicotiana benthamiana leaves.(c) Pull-down assay shows that the interaction of MdSPL1 and MdWRKY24 in in vitro.(d) EMSA assays show that the MdSPL1-MdWRKY24 interaction enhances the binding of MdSPL1 and MdWRKY24 to the 5 biotin probes.(e) and (f) LUC assays show that the MdSPL1-MdWRKY24 interaction enhances the repression of MdSPL1 and MdWRKY24 on MdSDH1.(g) Expression levels of MdSDH1, (h) sorbitol content, and (i) SDH enzyme activity in apple leaves with the overexpression of MdSPL1, MdWRKY24, and MdSPL1 + MdWRKY24.Values represent means ± SD (n = 3).Values with different letters are significantly different based on one-way ANOVA and Tukey's test (P < 0.05).

Figure 7 .
Figure 7. MdSPL1 and MdWRKY24 coordinately repress MdGASA1 expression.(a) Schematic diagram of the MdGASA1 promoter.(b) Y1H assays identify the binding ability of MdSPL1 and MdWRKY24 on MdGASA1 promoter.(c) Schematic diagram of reporter and effector constructs.(d) The images of luciferase and relative LUC/REN activities between MdGASA1 promoter and MdSPL1 and MdWRKY24.(e) EMSA assays of MdSPL1 and MdWRKY24 proteins binding to MdGASA1 promoter.Mut, mutant probes.(f) EMSA assays show that the MdSPL1-MdWRKY24 interaction enhances the binding of MdSPL1 and MdWRKY24 to the 5 biotin probes.(g) LUC assays show that the MdSPL1-MdWRKY24 interaction enhances the repression of MdSPL1 and MdWRKY24 on MdGASA1.Values represent means ± SD (n = 3).Values with different letters are significantly different based on one-way ANOVA and Tukey's test (P < 0.05).

Figure 8 .
Figure 8. Silencing MdGASA1 inhibits cell elongation in apple.(a) Phenotype of TRV-MdGASA1 and TRV apple plants.(b) Relative expression levels of MdGASA1.Values represent means ± SD (n = 3).(c) Plant height, (d) internode number, and (e) internode length in TRV and TRV-MdGASA1 plants.(h) Cross sections of the internodes.C: cambium, X: xylem.(f) Cortex cell length, (g) cortex cell area, and (i) xylem size based on the cross sections of TRV-MdGASA1 and TRV plants.(k) Longitudinal sections of the internodes.(j) Epidermal cell length, (l) epidermal cell area, and (m) epidermal cell number based on the longitudinal sections of TRV-MdGASA1 and TRV plants.Values represent means ± SD (n = 10).Values with different letters are significantly different based on one-way ANOVA and Tukey's test (P < 0.05).

Figure 9 .
Figure 9. Proposed models illustrating how sorbitol mediates age-dependent changes in apple plant growth strategy through gibberellin signaling.
30-day-old 1y apple plants were used for low-light treatment.The plants were placed in a light incubator (25 • C, 16/8 h light/dark) for 40 days.The normal light condition is 180 μmol m −2 s −1 and low-light condition is 35 μmol m −2 s −1 .The fully expanded leaves were collected at 0, 5, 15, 20, 30, and 40 days after treatment for RT-qPCR analysis.